Methods for modifying vascular vessel walls

ABSTRACT

This invention relates in one aspect to the treatment of a vascular vessel with a biomaterial. The biomaterial can be a remodelable material that strengthens and/or supports the vessel walls. Additionally the biomaterial can include a variety of naturally occurring or added bioactive agents and/or viable cellular populations.

REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalPatent Application Ser. No. 60/633,798 filed Dec. 7, 2004 which isincorporated herein by reference in its entirety. This application isalso a continuation-in-part of U.S. patent application Ser. No.11/266,166 filed Nov. 3, 2005, which claims the benefit of priority ofU.S. Provisional Patent Application Ser. No. 60/624,775 filed Nov. 3,2004, each of which is hereby incorporated herein in its entirety.

BACKGROUND OF THE INVENTION

In general, the present invention relates to methods of treatingpatients suffering from defects or diseases of the vascular system. Morespecifically, the present invention is directed to methods for modifyingvascular vessel walls.

Vascular vessels, such as veins and arteries, are prone to a variety ofcardiovascular disorders, for example, weakened walls possibly caused byarteriosclerosis or infection; and aneurysms, e.g. saccular, fusiform,and dissecting aneurysms. In the absence of treatment, the risks forfurther damage or rupture of an aneurysm can be very high and highlylethal should it occur. However, treatments for these defects mayrequire surgical intervention, which also poses significant risks. Forexample, it is suggested that for certain aortic or venous aneurysms,that a portion of the vessel be resected and replaced with vasculargraft, which is typically taken from one of the patient's extremities.Removal of a vein or artery from a patient's leg or arm can be quitepainful for the patient even months after the initial surgery.Additionally, blood flow in that leg or arm is also compromised. Asecond surgical site also poses additional risks to infections and othercomplications. It has also been suggested to use a synthetic graftmaterial repair the defect. Complications can also arise using syntheticmaterials to replace segments of vascular vessels, for examplethrombosis, rejection or secondary infection are possible.

There is a continuing need for advancements in the relevant field,including improved and/or alternative treatment methods and devices. Thepresent invention is addressed to these needs.

SUMMARY

In various aspects, the present invention relates to the treatment ofpatients with vascular diseases and the use of implantable devicestherefor. While the actual nature of the invention covered herein canonly be determined with reference to the claims appended hereto, certainforms and features, which are characteristic of the preferredembodiments disclosed herein, are described briefly as follows.

In one form, the present invention provides methods for modifying avascular vessel that has a defect. The methods can include accessing atreatment site proximate to the defect; and introducing a remodelablebiomaterial into the vessel wall or external of the vessel wall at thetreatment site. For example such methods can include treating a vasculardefect by reinforcing or remodeling the vessel wall. The vessel wall canbe reinforced by partly or fully encircling the vessel with a cuffformed of the biomaterial. In certain embodiments the cuff is formed ofa three dimensional construct of the biomaterial. In other embodiments,the cuff is formed from a fluidized form of the biomaterial, which hasgelled or solidified either prior to or after implantation in thepatient. The biomaterial can comprise a collagen-based material such asan extracellular matrix material. Submucosa tissue or another similarcollagenous layer can be used. Additionally, the biomaterial can includeadded or retained bioactive agents and/or viable cells. The biomaterialcan serve as a matrix to retain the bioactive agents and support aremodeling process. Other examples of treatment methods includeimplanting or introducing a biomaterial that can shrink in vivo. Thispropensity to shrink can be used to support and/or modify the vesselwalls.

In another form, the present invention provides methods for treating apatient with a vascular defect. The method comprises introducing adelivery device through the patient's vasculature to a treatment siteproximate to the defect; and delivering a remodelable biomaterial intoor external of a wall of the vessel, or a combination thereof. Incertain embodiments, the delivery device can be a needle or a cannulateddelivery device. The delivery device can be introduced in to thepatient's vascular, e.g., intra-lumenally (intravascularly), at a siteeither adjacent to or remote from the vascular defect. In otherembodiments, the delivery device can be introduced exo-lumenally(exovascularly). The biomaterial can be an injectable compositioncontaining a flowable mass of submocosa or other ECM derived material.The injectable biomaterial can be allowed or induced to gel or otherwiseharden upon injection into the treatment site.

In another form the present invention provides methods for treating avascular vessel in a patient. The method comprises introducing aremodelable biomaterial into or externally of a wall of the vessel in aregion proximate to aneurysm or wall defect.

The method can include using a three dimensional construct of thebiomaterial of a fluidized form of the biomaterial. In ether form, thebiomaterial can include a variety of added or naturally occurringbioactive agents and viable cells.

Further objects, features, aspects, forms, advantages and benefits shallbecome apparent from the description and drawings contained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides illustrations of cross-sections of a vein and an artery.

FIG. 2 is a cross-sectional view of a vascular vessel exhibiting onemanifestation of a defect.

FIG. 3 is a cross-sectional view of a remodeled vascular vessel with aband of biomaterial external to the vessel wall in accordance with thepresent invention.

FIG. 4 is one embodiment of a construct formed of a sheet of abiomaterial in accordance with the present invention.

FIG. 5 illustrates the positioning of the prosthesis of FIG. 4 about avessel in accordance with the present invention.

FIG. 6 is an illustration of an alternative embodiment of a constructformed of a sheet of a biomaterial in accordance with the presentinvention.

FIG. 7 illustrates the positioning of the prosthesis of FIG. 6 about avessel in accordance with the present invention.

FIG. 8 provides an illustration of a tissue volume containing a vascularvessel and in which an injected mass of biomaterial forms a cuff aroundthe vessel.

FIG. 9 provides a cross-sectional view illustrating the injection of amass of material to surround a vessel in the region of a defect.

FIG. 10 provides a cross-sectional view illustrating the injection of amaterial into the vessel wall in the region of a defect.

DETAILED DESCRIPTION

For the purposes of promoting an understanding of the invention, certaintreatment methods, prosthesis devices, and materials will be discussed.It will nevertheless be understood that no limitation of the scope ofthe invention is thereby intended. Any alterations and furthermodifications in the described materials, prostheses, and treatmentmethods, and any further applications of the principles of the inventionas described herein, are contemplated as would normally occur to oneskilled in the art to which the invent relates.

The present invention provides methods, devices, and materials fortreating vascular vessels at sites associated with vascular valves, orat sites other than those associated with vascular valves. Vascularvessels, including veins and arteries, are prone to a variety ofdiseases and defects that affect the function, strength, and integrityof the vessels in regions associated with valves within the vessels, orin other regions. Weakened vascular vessel walls can, for example, becaused by arteriosclerosis or infection; and aneurysms, e.g. saccular,fusiform, and dissecting aneurysms. The treatment according to thepresent invention can include introducing a biomaterial into a treatmentsite located in tissue adjacent the vessel and/or in the vessel's walls.The biomaterial provides a benefit to the vascular vessels, for exampleto treat defects and diseases as described more fully herein. Thematerials and methods described herein are advantageous for treatment ofdisorders associated with venous valves, particularly those occurringwithin limbs such as legs. Further, the biomaterial can be a remodelablematerial and may include one or more bioactive substances derived fromthe source of the biomaterial and/or added to the biomaterial. Thebiomaterial can also be seeded with or contain viable cells.

For the purposes of certain aspects of the descriptions herein, FIG. 1illustrates a cross-sectional view of a normal vein and an artery.Artery 10 is composed of three coats: an internal or endothelial coat(tunica intima) 12; a middle muscular coat (tunica media) 14, and anexternal connective tissue coating (tunica adventitia) 16. Typically,the two inner coats 12 and 14 together are very easily separated fromthe external coating. The vein 18 is also composed of threecoats—internal, middle, and external coats 20, 22, and 24, respectively.A significant structural difference between veins and arteries is thecomparative weakness of the middle coat of the vein. Because of this,the veins do not stand open when divided as the arteries do.Consequently, a vein tends to collapse without an internal fluid orfluid pressure, while a healthy artery substantially retains its naturalshape and relative dimension regardless of whether or not it isconducting blood (or other fluid). Additionally, many veins, includingthe larger veins in the lower extremities, contain multiple valves toprevent the reflux or retrograde flow of blood away from the heart andthereby facilitate the return of blood to the heart.

“Remodeling” as used herein generally refers to the production of hosttissue that replaces implanted material. The development of host tissuecan occur at a functional rate about equal to the rate of biodegradationof the implanted material, resulting in a replacement of the implantedmaterial by host-generated tissue, including for example structuralproteins such as collagen, and cells.

In certain embodiments, the present invention provides a treatment forvascular vessels using a prosthesis formed of a biomaterial-containingconstruct. It is preferred to use a remodelable biomaterial that canalso serve as a biocompatible scaffold with the ability to remodel hosttissue. Accordingly, a naturally occurring biomaterial is highlydesirable. In this regard, biomaterials for use in the present inventioncan be obtained as a purified collagen-containing matrix structure.

One such collagen-containing biomaterial is extracellular matrix (ECM).Preferred are naturally derived collagenous ECMs isolated from suitableanimal or human tissue sources. Suitable extracellular matrix materialsinclude, for instance, submucosa (including for example small intestinalsubmucosa, stomach submucosa, urinary bladder submucosa, or uterinesubmucosa, each of these isolated from juvenile or adult animals), renalcapsule membrane, amnion, dura mater, pericardium, serosa, peritoneum orbasement membrane materials, including liver basement membrane orepithelial basement membrane materials. These materials may be isolatedand used as intact natural sheet forms, or reconstituted collagen layersincluding collagen derived from these materials and/or other collagenousmaterials may be used. For additional information as to submucosamaterials useful in the present invention, and their isolation andtreatment, reference can be made to U.S. Pat. Nos. 4,902,508, 5,554,389,5,993,844, 6,206,931, and 6,099,567. Renal capsule membrane can also beobtained from warm-blooded vertebrates, as described more particularlyin International Patent Application serial No. PCT/US02/20499 filed Jun.28, 2002, published Jan. 9, 2003 as WO03002165.

The biomaterial can retain growth factors or other bioactive componentsnative to a source tissue. For example, submucosa or other ECM materialsmay include one or more growth factors such as basic fibroblast growthfactor (FGF-2), transforming growth factor beta (TGF-beta), epidermalgrowth factor (EGF), and/or platelet derived growth factor (PDGF). Aswell, submucosa or other ECM tissue used in the invention may includeother biological materials such as heparin, heparin sulfate, hyaluronicacid, fibronectin and the like. Thus, generally speaking, the submucosaor other ECM material may include a bioactive component that induces,directly or indirectly, a cellular response such as a change in cellmorphology, proliferation, growth, protein or gene expression.

The biomaterial can be used alone, or in combination with one or moreadded pharmacologic agents, such as physiologically compatible minerals,growth factors (including vascular endothelial growth factors),antibiotics, chemotherapeutic agents, antigens, antibodies, geneticmaterial, enzymes and hormones and the like.

In certain forms, the biomaterial of the invention can also be used incombination with other nutrients which support the growth of cells, e.g.eukaryotic cells such as endothelial (including non-keratinized orkeratinized epithelial cells), fibroblastic cells, smooth muscle cells,cardiac muscle cells, multipotent progenitor or stem cells, pericytes,or any other suitable cell type (see, e.g. International Publication No.WO 96/24661 dated 15 Aug. 1996, publishing International Application No.PCT/US96/01842 filed 9 Feb. 1996). Such cells may optionally be includedin the biomaterial construct or composition, for example being seededupon or incorporated within the biomaterial construct or composition.The submucosa or other biomaterial substrate composition can beeffective to support the proliferation and/or differentiation ofmammalian cells, including human cells. Still further, the inventivemethods herein may use a biomaterial that serves as a matrix that cansupport and produce genetically modified cells, (see, e.g.,International Publication No. WO 96/25179 dated 22 Aug. 1996, publishingInternational Application No. PCT/US96/02136 filed 16 Feb. 1996; andInternational Publication No. WO 95/22611 dated 24 Aug. 1995, publishingInternational Application No. PCT/US95/02251 filed 21 Feb. 1995). Suchcompositions for genetically modifying cells can include an ECM such assubmucosa or another collagenous biomaterial as a three dimensionalconstruct or a fluidized or flowable material in combination with anucleic acid molecule containing a sequence to be expressed in cells,e.g. a recombinant vector such as a plasmid containing a nucleic acidsequence with which in vitro or in vivo target cells are to begenetically modified.

It some forms of practicing the invention, the biomaterial according tothe present invention is substantially free of any antiviral agents orany antimicrobial-type agents, which can affect the biochemistry of thebiomaterial and its efficacy upon implantation. One method of treatingtissue material is to rinse the delaminated tissue in saline and soak itin an antimicrobial agent, for example, as disclosed in U.S. Pat. Nos.4,956,178 and 6,666,892. While such techniques can optionally bepracticed with isolated collagenous ECMs such as submucosa in thepresent invention, preferred embodiments avoid the use of antimicrobialagents and the like, which not only can affect the biochemistry of thecollagenous biomaterial but also can be unnecessarily introduced intothe tissues of the patient.

Irrespective of the origin of the biomaterial, a prosthesis for use inthe present invention can be made thicker by making multilaminateconstructs, for example SIS constructs or materials as described in U.S.Pat. Nos. 5,968,096; 5,955,110; 5,885,619; and 5,711,969; thedisclosures of which are entirely and expressly incorporated byreference. The layers of the laminated construct can be bonded togethervia a crosslinking agent, a bonding agent, dehydrothermal bonding and/orby use of biotissue welding technique(s), or other suitable means.

In certain embodiments, the ECM or other biomaterial can be initiallyprocessed to provide a predetermined, three-dimensional shape orconstruct, which will be implanted into the patient as a prosthesis andwill substantially retain its shape during remodeling or replacement ofthe graft with endogenous tissues. Illustratively, in embodimentswherein a sheet-form collagenous biomaterial is provided partially orcompletely encircling a vein or artery, the biomaterial can be processedto have the shape of a complete or partial cylinder configured to fitaround and contact the external walls of the vessel about which theconstruct is to be received. Processing to provide such shapes caninclude configuring the ECM or other biomaterial to a desired shapewhile fully or partially hydrated, and dehydrating the material while insuch shape. Such dehydrating may for example be conducted by air drying,lyophilization, vacuum pressing, or other suitable techniques.

For treatment of blood containing vessels in accordance with the presentinvention, and particularly for situations in which exposure of anamount of the biomaterial to the interior of the vessel is needed or arisk, the biomaterial can be treated to reduce any thrombogeniccharacter that it may have. In this regard, an antithrombotic agent,such as, heparin or a heparin derivative may be bound to thebiomaterial, construct or a prosthesis formed from the construct by anysuitable method including physical, ionic, or covalent bonding. Forexample, this may be accomplished for example by applying solution ofheparin or a heparin derivative to the biomaterial's surface or bydipping the biomaterial in the solution. In one embodiment, heparin isbound to the biomaterial using a suitable crosslinking agent such as apolyepoxide or carbodiimide cross linking agent such as1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC). Inmulti-layer constructs, heparin or other agents can be applied to thelayers individually before incorporation of the layer into theconstruct, after the layers are incorporated into the construct (e.g.coating a luminal surface of an inner tubular layer), or both. Heparincan also be applied using a benzalkonium heparin (BA-Hep) isopropylalcohol solution. This procedure treats the collagen with an ionicallybound BA-Hep complex. Other coating, bonding, and attachment procedures,which are known in the art, can also be used. As well, in the case offlowable (e.g. injectable) biomaterials, heparin or one or more otheranti-thrombogenic agents may be incorporated into the formulationutilized in soluble form and/or bound to any suspended particulatebiomaterial within the formulation.

The present invention provides for the treatment of vascular defectsassociated with vascular valves, in certain of its embodiments. Inpreferred embodiments, the present invention provides for the treatmentof vascular defects associated with venous valves. Illustratively, FIG.2 shows a cross-sectional view of a portion of vascular vessel 30 havinga defect generally depicted as distended wall 32. Typically, althoughnot required, the distended wall is indicative of a weakened vascularwall. The weakened wall portion can present a serious risk of rupturethat can lead to death and/or interruption of blood flow and function ofa nearby valve.

In certain embodiments of the invention, such a diseased or defectiveregion of the vascular vessel is identified in the patient and isaccessed for treatment. This access can be provided, for example, usingsurgical cut-down procedures, or using minimally invasive proceduressuch as percutaneous and/or endoscopic techniques. Typically the defect(aneurysm, fistula or the like) is also identified/or confirmed and thetreatment methods tailored accordingly. For treatment methods of theinvention including the implantation of biomaterials external of thevascular vessel, exposure of or other access to the entire externalcircumference of the vascular vessel, or only to one or multiple pointsaround the circumference of the vessel, or between two adjacent vessels,may be utilized.

In other treatment methods, which use a fluidized biomaterial, access tothe treatment site can be accomplished by introducing a needle or othercannulated delivery device subcutaneously or intravascularly to deliverthe biomaterial to the treatment site. When accessed intravascularly thedelivery device can be guided through a portion of the patient'svascular system using known imaging techniques.

FIG. 3 is an illustration of a vascular vessel 50 and including a band52 of biomaterial external to the vessel wall 54 resulting from one formof treatment according to one form of treatment according to the presentinvention. Band 52 of biomaterial can be used to address the defect ordisorder in the vascular vessel. In one form, band 52 can providesupport for the vessel wall 54 or cover a nick or tear in the vessel toname but two applications. Band 52 can be formed by wrapping a constructof the biomaterial about the outer surface of vessel 50, such asdiscussed below for FIGS. 4 and 6. Alternatively, band 52 can be formedby applying a fluidized biomaterial adjacent to the external surface ofvessel 50.external connective coating of the vessel.

FIG. 4 is an illustration of one embodiment of a construct 70 to form aprosthesis in accordance with the present invention. Construct 70 isformed of a remodelable biomaterial, and in one particular embodimentconstruct 70 is formed from submucosa. Construct 70 is illustrated as anelongated, planar construct, preferably in the form of a flexible sheet.Construct 70 can be composed of a single layer or of several layers as alaminate of the remodelable biomaterial. Construct 70 can be sized andshaped either prior to or during surgery to approximate at least aportion of the external circumference of a targeted vessel. Further,construct 70 can be prepared to include one or more bioactive agentsand/or cellular populations depending upon the desired treatment regime.

In use, once the treatment site has been accessed sufficiently,construct 70 is wrapped about the external circumference of the targetvessel overlaying the treatment site. Construct 70 can partly orcompletely encircle the vessel. The first and second ends 72 and 74 ofthe construct 70 can be sutured or secured to the external circumferenceof the vessel wall in certain embodiments. In other embodiments,construct 70 is sized and formed such that the two opposite ends 72 and74 touch or overlap each other. The two edges can be secured togetherusing conventional means including suturing to each other (and/or to thevessel wall), tissue welding (see e.g. U.S. Pat. No. 5,156,613), and/orbonding agents such as fibrin glue, or other suitable techniques.

In this regard, FIG. 5 provides an illustration of construct 70 disposedabout the external wall (or connective tissue coating) of a vessel 76.Construct 70 can be secured using sutures. The illustrated construct 70can be used to remodel and/or support the vessel wall against initial orcontinued distention. Further, construct 70 can be used to reinforce orrepair the vessel wall so that the vessel can withstand or exceedanticipated dynamic fluid pressure to which it may be subjected during apatient's normal level of physical activity.

FIG. 6 illustrates an alternative embodiment of a construct 80 for usein the present invention. Construct 80 includes a slot 82 formed at itsfirst end and a corresponding tab 84 extending from a second end. Inuse, tab 84 can be inserted into slot 82 to provide a generally circularcuff or band that can be placed around a vein or artery. Additionally,the ends, including tab 84, can optionally be secured together asdiscussed above for construct 70.

FIG. 7 provides an illustration of construct 80 received and securedaround a vascular vessel 86 in the region of a treatment site. Inparticular, as with the construct 70 illustrated in FIG. 5, theillustrated construct 80 covers the area of the vein wall correspondingto the base of the leaflets. This positioning can be used to support thevein wall against initial or continued distention and/or to re-shape thevessel to increase the burst strength improve elasticity of the vessel.It will also be understood that construct 80 can include plurality oftabs and corresponding number of slots to facilitate securing theconstruct around a vein or other vascular vessel.

Although FIG. 5 and FIG. 7 show the constructs 70 and 80 positionedabout the exterior circumference of the vessel proximate to the base ofthe leaflets, it will be understood that other regions may also betreated in accordance with the invention. The prosthesis can bepositioned directly at or about the base of the leaflets or below thebase of the leaflets, i.e., further from the heart. Alternatively, theprosthesis can be positioned above the base of the leaflets, i.e.,closer to the heart. In other embodiments, two or more prostheses can beused, for example, one placed above and another placed below the base ofthe leaflets. In still other embodiments, the prosthesis can include areinforcing clamp or clip received around the construct 70 or 80. Thereinforcing clamp or clip can provide further support and compressagainst the external side walls of vessel.

The thickness of the construct(s) to form the prosthesis can be selecteddepending upon the tissue or biomaterial used, its intended use, and/ora prescribed treatment regime. In preferred embodiments for thetreatment of veins and arteries, the biomaterial can be provided in athickness of between about 50 and about 500 microns. It will beunderstood that the thickness of the construct/prosthesis can also bevaried or, in particular, increased by laminating two or more layers ofa biomaterial onto one another. It will also be understood that otherconfigurations of the prosthesis such as the tissue constructs describedin U.S. patent application Ser. No. 10/068,212 filed Feb. 6, 2002, canbe used.

As discussed above, when the vessel exhibits a defect such as thatdescribed above for venous valvular dysfunction, the construct orprosthesis can serve to modify the shape of the vessel in the region ofthe leaflet bases and/or in regions immediately upstream or downstreamof the leaflet bases. This, in turn, can reduce the interior dimension,in particular the interior diameter of the vessel.

A sufficient amount of the remodelable biomaterial is used to treat thevessel. In certain embodiments, a sufficient amount of material is usedto encircle greater than about 20% of the circumference of the vessel;more preferably, a sufficient amount of the remodelable biomaterial isused to encircle greater than about 60% of the vessel; still morepreferable, greater than about 80% of the vessel. In certainembodiments, an effective amount of the biomaterial is implanted aboutthe vessel to reinforce the vessel wall, and/or to modify the diameteror shape of the vessel wall.

In some embodiments of the invention, the biomaterial is selected so asto retract or shrink after implantation in vivo. For instance, as aremodelable material retracts upon remodeling, it can press against orcompress the periphery of the vessel. The remodelable material can beprepared to shrink at varying rates and to varying extents as desired.The shrinkage can also be controlled by a judicious selection of thesource of the biomaterial and its preparation. For example thebiomaterial can include a two or more layers, which are layers can beobtained from different tissue or obtained from the same tissue type butprepared differently. Alternatively or in addition, the selectedbiomaterial may exhibit anisotropic shrinkage, e.g. upon remodeling withhost tissue. Consequently, the biomaterial shrinks to a greater extentin a first direction while exhibiting relatively less shrinkage in asecond direction. Two or more layers of such a biomaterial either aloneor in conjunction with other layers can be laminated such that theanisotropic shrinkage affects of the layers either complements eachother, e.g. as in multilaminate materials having the layers orientedwith their directions of maximal shrinkage generally aligned, orinterferes with or modifies each other, e.g. as in multilaminatematerials having the layers oriented with their directions of maximalshrinkage non-aligned or generally transverse to one another.

The biomaterial can also be crosslinked to vary and/or control theextent and/or rate of shrinkage. Increasing the amount (or number) ofcrosslinkages within the biomaterial or between two or more layers ofthe construct can be used to decrease the relative amount of shrinkage.However, crosslinkages within the biomaterial may also effect itsremodelability. Consequently, in applications where tissue remodeling isdesired, the biomaterial may substantially retain its native level ofcrosslinking, or the amount of added crosslinkages within thebiomaterial can be judiciously selected depending upon the desiredtreatment regime. In many cases, the biomaterial will exhibitremodelable properties such that the remodeling process occurs over thecourse of several days or several weeks. In preferred embodiments, theremodeling process occurs within a matter of about 5 days to about 12weeks.

For use in the present invention, introduced crosslinking of thebiomaterial may be achieved by photo-crosslinking techniques, bychemical crosslinkers, or by protein crosslinking induced by dehydrationor other means. Chemical crosslinkers that may be used include forexample aldehydes such as glutaraldehydes, diimides such ascarbodiimides, e.g., 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride, ribose or other sugars, acyl-azide,sulfo-N-hydroxysuccinamide, or polyepoxide compounds, including forexample polyglycidyl ethers such as ethyleneglycol diglycidyl ether,available under the trade name DENACOL EX810 from Nagese Chemical Co.,Osaka, Japan, and glycerol polyglycerol ether available under the tradename DENACOL EX 313 also from Nagese Chemical Co. Typically, when used,polyglycerol ethers or other polyepoxide compounds will have from 2 toabout 10 epoxide groups per molecule.

When a laminate of material is used in the present invention, the layersof the laminate can be additionally crosslinked to bond multiplesubmucosa layers to one another. Thus, additional crosslinking may beadded to individual submucosa layers prior to bonding to one another,during bonding to one another, and/or after bonding to one another.

In some embodiments of the invention, the prosthesis is prepared using atwo component bonding agent such as fibrin glue (e.g., having thrombinand fibrinogen as separate components). To prepare such prostheses,subsequent layers are added after coating the previously-applied layerwith a first component of the bonding agent (e.g., thrombin) and coatinga layer to be applied with a second component of the bonding agent(e.g., fibrinogen). Thereafter, the layer to be applied is positionedover the previously-applied layer so as to bring the two bondingcomponents into contact, thus causing the curing process to begin. Thisprocess can be repeated for any and all additional layers in a laminatedconstruct. Additionally this process can be used to bond the ends of aprosthesis together in vivo.

In another mode of treatment, an injectable or otherwise flowablebiocompatible material can be introduced in the patient to treat avascular vessel site associated with a valve. For example, such acomposition can be introduced into the vessel wall, e.g. into or inbetween one or more coatings of the vessel wall, and/or can beintroduced into an external region surrounding a vessel wall, e.g. incontact with the external connective tissue coating (tunica adventitia)of an vein or artery. The injectable or flowable composition can beintroduced at a single site or at a plurality of sites within the wallof a vascular vessel and/or about the external periphery of the vascularvessel. Relatively non-invasive modes of introduction will beadvantageous, e.g. involving the delivery of the flowable compositionthrough a cannulated device such as a needle or catheter.

In certain embodiments of the invention, an effective amount of aflowable composition is injected or otherwise introduced so as toprovide support for and/or increase the strength of vessel walls. Theintroduced composition can include one or more bioactive agents orviable cellular populations, or combinations of these. Non-limitingexamples of bioactive agents are listed above. Preferably the bioactiveagents are effective to promote tissue growth.

While other biocompatible materials that can provide bulk mass toreinforce or modify the shape of the vessel walls may be used, theinjectable or flowable composition for use in the invention desirablycomprises an ECM composition. For example, fluidized submucosa can beprepared as described in U.S. Pat. Nos. 5,275,826 and 6,444,229.Fluidized or otherwise flowable ECM compositions may also be prepared asdescribed in co-pending International Patent Application No.PCT/US04/27557 filed Aug. 25, 2004 and entitled Graft MaterialsContaining Bioactive Substances, and Methods for Their Manufacture. Inthis regard, the flowable compositions of the invention can be preparedfrom an isolated ECM material, for example one of those listed above.The ECM material is used to prepare a solubilized mixture includingcomponents of the material. This can be achieved by digestion of the ECMmaterial in an acidic or basic medium and/or by contact with anappropriate enzyme or combination of enzymes.

The ECM material can be reduced to particulate form to aid in thedigestion step. This can be achieved by tearing, cutting, grinding orshearing the isolated ECM material. Illustratively, shearing may beconducted in a fluid medium, and grinding may be conducted with thematerial in a frozen state. For example, the material can be contactedwith liquid nitrogen to freeze it for purposes of facilitating grindinginto powder form. Such techniques can involve freezing and pulverizingsubmucosa under liquid nitrogen in an industrial blender.

Any suitable enzyme may be used for an enzymatic digestion step. Suchenzymes include for example serine proteases, aspartyl proteases, andmatrix metalloproteases. The concentration of the enzyme can be adjustedbased on the specific enzyme used, the amount of submucosa to bedigested, the duration of the digestion, the temperature of thereaction, and the desired properties of the final product. In oneembodiment about 0.1% to about 0.2% of enzyme (pepsin, for example) isused and the digestion is conducted under cooled conditions for a periodof time sufficient to substantially digest the ECM material. Thedigestion can be conducted at any suitable temperature, withtemperatures ranging from 4-37° C. being preferred. Likewise, anysuitable duration of digestion can be used, such durations typicallyfalling in the range of about 2-180 hours. The ratio of theconcentration of ECM material (hydrated) to total enzyme usually rangesfrom about 25 to about 125 and more typically the ratio is about 50, andthe digestion is conducted at 4° C. for 24-72 hours. When an enzyme isused to aid in the digestion, the digestion will be performed at a pH atwhich the enzyme is active and more advantageously at a pH at which theenzyme is optimally active. Illustratively, pepsin exhibits optimalactivity at pH's in the range of about 2-4.

The enzymes or other disruptive agents used to solubilize the ECMmaterial can be removed or inactivated before or during the gellingprocess so as not to compromise gel formation or subsequent gelstability. Also, any disruptive agent, particularly enzymes, thatremains present and active during storage of the tissue will potentiallychange the composition and potentially the gelling characteristics ofthe solution. Enzymes, such as pepsin, can be inactivated with proteaseinhibitors, a shift to neutral pH, a drop in temperature below 0° C.,heat inactivation or through the removal of the enzyme by fractionation.A combination of these methods can be utilized to stop digestion of theECM material at a predetermined endpoint, for example the ECM materialcan be immediately frozen and later fractionated to limit digestion.

The ECM material is enzymatically digested for a sufficient time toproduce a hydrolysate of ECM components. The ECM can be treated with oneenzyme or with a mixture of enzymes to hydrolyze the structuralcomponents of the material and prepare a hydrolysate having multiplehydrolyzed components of reduced molecular weight. The length ofdigestion time is varied depending on the application, and the digestioncan be extended to completely solubilize the ECM material. In some modesof operation, the ECM material will be treated sufficiently to partiallysolubilize the material to produce a digest composition comprisinghydrolyzed ECM components and nonhydrolyzed ECM components. The digestcomposition can then optionally be further processed to remove at leastsome of the nonhydrolyzed components. For example, the nonhydrolyzedcomponents can be separated from the hydrolyzed portions bycentrifugation, filtration, or other separation techniques known in theart.

Preferred gel compositions of the present invention are prepared fromenzymatically digested vertebrate ECM material that has beenfractionated under acidic conditions, for example including pH rangingfrom about 2 to less than 7, especially to remove low molecular weightcomponents. Typically, the ECM hydrolysate is fractionated by dialysisagainst a solution or other aqueous medium having an acidic pH, e.g. apH ranging from about 2 to about 5, more desirably greater than 3 andless than 7. In addition to fractionating the hydrolysate under acidicconditions, the ECM hydrolysate is typically fractionated underconditions of low ionic strength with minimal concentrations of saltssuch as those usually found in standard buffers such as PBS (i.e. NaCl,KCl, Na₂HPO₄, or KH₂PO₄) that can pass through the dialysis membrane andinto the hydrolysate. Such fractionation conditions work to reduce theionic strength of the ECM hydrolysate and thereby provide enhanced gelforming characteristics.

The hydrolysate solution produced by enzymatic digestion of the ECMmaterial has a characteristic ratio of protein to carbohydrate. Theratio of protein to carbohydrate in the hydrolysate is determined by theenzyme utilized in the digestion step and by the duration of thedigestion. The ratio may be similar to or may be substantially differentfrom the protein to carbohydrate ratio of the undigested ECM tissue. Forexample, digestion of vertebrate ECM material with a protease such aspepsin, followed by dialysis, will form a fractionated ECM hydrolysatehaving a lower protein to carbohydrate ratio relative to the originalECM material.

Flowable ECM compositions capable of forming shape retaining gels can beused in the present invention. Such ECM compositions can be preparedfrom ECM material that has been enzymatically digested and fractionatedunder acidic conditions to form an ECM hydrolysate that has a protein tocarbohydrate ratio different than that of the original ECM material.Such fractionation can be achieved entirely or at least in part bydialysis. The molecular weight cut off of the ECM components to beincluded in the gellable material is selected based on the desiredproperties of the gel. Typically the molecular weight cutoff of thedialysis membrane (the molecular weight above which the membrane willprevent passage of molecules) is within in the range of about 2000 toabout 10000 Dalton, and more preferably from about 3500 to about 5000Dalton.

In certain forms of the gellable ECM composition, apart from thepotential removal of undigested ECM components after the digestion stepand any controlled fractionation to remove low molecular weightcomponents as discussed above, the ECM hydrolysate is processed so as toavoid any substantial further physical separation of the ECM components.For example, when a more concentrated ECM hydrolysate material isdesired, this can be accomplished by removing water from the system(e.g. by evaporation or lyophilization) as opposed to using conventional“salting out”/centrifugation techniques that would demonstratesignificant selectivity in precipitating and isolating collagen, leavingbehind amounts of other desired ECM components. Thus, in certainembodiments of the invention, solubilized ECM components of the ECMhydrolysate remain substantially unfractionated, or remain substantiallyunfractionated above a predetermined molecular weight cutoff such asthat used in the dialysis membrane, e.g. above a given value in therange of about 2000 to 10000 Dalton, more preferably about 3500 to about5000 Dalton.

Vertebrate ECM material can be stored frozen (e.g. at about −20 to about−80° C.) in either its solid, comminuted or enzymatically digestedforms, or the material can be stored after being hydrolyzed andfractionated. The ECM material can be stored in solvents that maintainthe collagen in its native form and solubility. For example, onesuitable storage solvent is 0.01 M acetic acid, however other acids canbe substituted, such as 0.01N HCl. In one form, the fractionated ECMhydrolysate can be dried (by lyophilization, for example) and stored ina dehydrated/lyophilized state. The dried form can be rehydrated toprepare a flowable ECM composition capable of forming a gel.

In accordance with one embodiment, the fractionated ECM hydrolysate orother flowable ECM composition will exhibit the capacity to gel uponadjusting the pH of a relatively more acidic aqueous medium containingit to about 5 to about 9, more preferably about 6.6 to about 8.0, andtypically about 7.2 to about 7.8, thus inducing fibrillogenesis andmatrix gel assembly. In one embodiment, the pH of the fractionatedhydrolysate is adjusted by the addition of a buffer that does not leavea toxic residue, and has a physiological ion concentration and thecapacity to hold physiological pH. Examples of suitable buffers includePBS, HEPES, and DMEM. Illustratively, the pH of the fractionated ECMhydrolysate can be raised by the addition of a buffered NaOH solution to6.6 to 8.0, more preferably 7.2 to 7.8, to facilitate the formation ofan ECM-containing gel. Any suitable concentration of NaOH solution canbe used for these purposes, for example including about 0.05 M to about0.5 M NaOH. In accordance with one embodiment, the ECM hydrolysate ismixed with a buffer and sufficient 0.25N NaOH is added to the mixture toachieve the desired pH.

The ionic strength of the ECM hydrolysate is believed to be important inmaintaining the fibers of collagen in a state that allows forfibrillogenesis and matrix gel assembly upon neutralization of thehydrolysate. Accordingly, if needed, the salt concentration of the ECMhydrolysate material can be reduced prior to neutralization of thehydrolysate. The neutralized hydrolysate can be caused to gel at anysuitable temperature, e.g. ranging from about 4° C. to about 40° C. Thetemperature will typically affect the gelling times, which may rangefrom 5 to 120 minutes at the higher gellation temperatures and 1 to 8hours at the lower gellation temperatures. Typically, the hydrolysatewill be effective to self-gel at elevated temperatures, for example atabout 37° C. In this regard, preferred neutralized ECM hydrolysates willbe effective to gel in less than about ninety minutes at 37° C., forexample approximately thirty to ninety minutes at 37° C.

Additional components can be added to the ECM hydrolysate compositionbefore, during or after forming the gel. For example, proteinscarbohydrates, growth factors, therapeutics, bioactive agents, nucleicacids, cells or pharmaceuticals can be added. In certain embodiments,such materials are added prior to formation of the gel. This may beaccomplished for example by forming a dry mixture of a powdered ECMhydrolysate with the additional component(s), and then reconstitutingand gelling the mixture, or by incorporating the additional component(s)into an aqueous, ungelled composition of the ECM hydrolysate before,during (e.g. with) or after addition of the neutralization agent. Theadditional component(s) can also be added to the formed ECM gel, e.g. byinfusing or mixing the component(s) into the gel and/or coating themonto the gel.

In one embodiment of the invention, a particulate ECM material will beadded to the ECM hydrolysate composition, which will then beincorporated in the formed gel. Such particulate ECM materials can beprepared by cutting, tearing, grinding or otherwise comminuting an ECMstarting material. For example, a particulate ECM material having anaverage particle size of about 50 microns to about 500 microns may beincluded in the gellable ECM hydrolysate, more preferably about 100microns to about 400 microns. The ECM particulate can be added in anysuitable amount relative to the hydrolysate, with preferred ECMparticulate to ECM hydrolysate weight ratios (based on dry solids) beingabout 0.1:1 to about 200:1, more preferably in the range of 1:1 to about100:1. The inclusion of such ECM particulates in the ultimate gel canserve to provide additional material that can function to providebioactivity to the gel (e.g. itself including FGF-2 and/or other growthfactors or bioactive substances as discussed herein) and/or serve asscaffolding material for tissue ingrowth.

In certain embodiments, an ECM hydrolysate material to be used in theinvention will exhibit an injectable character and also incorporate anECM particulate material. In these embodiments, the ECM particulatematerial can be included at a size and in an amount that effectivelyretains an injectable character to the hydrolysate composition, forexample by injection through a needle having a size in the range of 18to 31 gauge (internal diameters of 0.047 inches to about 0.004 inches).In this fashion, non-invasive procedures for tissue augmentation will beprovided, which in preferred cases will involve the injection of anungelled ECM hydrolysate containing suspended ECM particles at arelatively lower (e.g. room) temperature, which will be promoted to forma gelled composition when injected into a patient and thereby brought tophysiologic temperature (about 37° C.).

In certain embodiments, flowable ECM compositions to be used in theinvention may be disinfected by contacting an aqueous medium includingECM hydrolysate components with an oxidizing disinfectant. This mode ofdisinfection provides an improved ability to recover a disinfected ECMhydrolysate that exhibits the capacity to form beneficial gels. Incertain preparative methods, an aqueous medium containing ECMhydrolysate components can disinfected by providing a peroxydisinfectant in the aqueous medium. This can be advantageously achievedusing dialysis to deliver the peroxy disinfectant into and/or to removethe peroxy disinfectant from the aqueous medium containing thehydrolysate. In certain dinsinfection techniques, an aqueous mediumcontaining the ECM hydrolysate is dialyzed against an aqueous mediumcontaining the peroxy disinfectant to deliver the disinfectant intocontact with the ECM hydrolysate, and then is dialyzed against anappropriate aqueous medium (e.g. an acidic aqueous medium) to at leastsubstantially remove the peroxy disinfectant from the ECM hydrolysate.During this dialysis step, the peroxy compound passes through thedialysis membrane and into the ECM hydrolysate, and contacts ECMcomponents for a sufficient period of time to disinfect the ECMcomponents of the hydrolysate. In this regard, typical contact timeswill range from about 0.5 hours to about 8 hours and more typicallyabout 1 hour to about 4 hours. The period of contact will be sufficientto substantially disinfect the digest, including the removal ofendotoxins and inactivation of virus material present. The removal ofthe peroxy disinfectant by dialysis may likewise be conducted over anysuitable period of time, for example having a duration of about 4 toabout 180 hours, more typically about 24 to about 96 hours. In general,the disinfection step will desirably result in a disinfected ECMhydrolysate composition having sufficiently low levels of endotoxins,viral burdens, and other contaminant materials to render it suitable formedical use. Endotoxin levels below about 2 endotoxin units (EUs) pergram (dry weight) are preferred, more preferably below about 1 EU pergram, as are virus levels below 100 plaque forming units per gram (dryweight), more preferably below 1 plaque forming unit per gram.

The aqueous ECM hydrolysate composition can be a substantiallyhomogeneous solution during the dialysis step for delivering theoxidizing disinfectant to the hydrolysate composition and/or during thedialysis step for removing the oxidizing disinfectant from thehydrolysate composition. Alternatively, the aqueous hydrolysatecomposition can include suspended ECM hydrolysate particles, optionallyin combination with some dissolved ECM hydrolysate components, duringeither or both of the oxidizing disinfectant delivery and removal steps.Dialysis processes in which at least some of the ECM hydrolysatecomponents are dissolved during the disinfectant delivery and/or removalsteps are preferred and those in which substantially all of the ECMhydrolysate components are dissolved are more preferred.

The disinfection step can be conducted at any suitable temperature, andwill typically be conducted between 0° C. and 37° C., more typicallybetween about 4° C. and about 15° C. During this step, the concentrationof the ECM hydrolysate solids in the aqueous medium can be in the rangeof about 2 mg/ml to about 200 mg/ml, and may vary somewhat through thecourse of the dialysis due to the migration of water through themembrane. In certain embodiments, a relatively unconcentrated digest isused, having a starting ECM solids level of about 5 mg/ml to about 15mg/ml. In other embodiments, a relatively concentrated ECM hydrolysateis used at the start of the disinfection step, for example having aconcentration of at least about 20 mg/ml and up to about 200 mg/ml, morepreferably at least about 100 mg/ml and up to about 200 mg/ml. It hasbeen found that the use of concentrated ECM hydrolysates during thisdisinfection processing results in an ultimate gel composition havinghigher gel strength than that obtained using similar processing with alower concentration ECM hydrolysate. Accordingly, processes whichinvolve the removal of amounts of water from the ECM hydrolysateresulting from the digestion prior to the disinfection processing stepare preferred. For example, such processes may include removing only aportion of the water (e.g. about 10% to about 98% by weight of the waterpresent) prior to the dialysis/disinfection step, or may includerendering the digest to a solid by drying the material by lyophilizationor otherwise, reconstituting the dried material in an aqueous medium,and then treating that aqueous medium with the dialysis/disinfectionstep.

In one mode of operation, the disinfection of the aqueous mediumcontaining the ECM hydrolysate can include adding the peroxy compound orother oxidizing disinfectant directly to the ECM hydrolysate, forexample being included in an aqueous medium used to reconstitute a driedECM hydrolysate or being added directly to an aqueous ECM hydrolysatecomposition. The disinfectant can then be allowed to contact the ECMhydrolysate for a sufficient period of time under suitable conditions(e.g. as described above) to disinfect the hydrolysate, and then removedfrom contact with the hydrolysate. In one embodiment, the oxidizingdisinfectant can then be removed using a dialysis procedure as discussedabove. In other embodiments, the disinfectant can be partially orcompletely removed using other techniques such as chromatographic or ionexchange techniques, or can be partially or completely decomposed tophysiologically acceptable components. For example, when using anoxidizing disinfectant containing hydrogen peroxide (e.g. hydrogenperoxide alone or a peracid such as peracetic acid), hydrogen peroxidecan be allowed or caused to decompose to water and oxygen, for examplein some embodiments including the use of agents that promote thedecomposition such as thermal energy or ionizing radiation, e.g.ultraviolet radiation.

In another mode of operation, the oxidizing disinfectant can bedelivered into the aqueous medium containing the ECM hydrolysate bydialysis and processed sufficiently to disinfect the hydrolysate (e.g.as described above), and then removed using other techniques such aschromatographic or ion exchange techniques in whole or in part, orallowed or caused to decompose in whole or in part as discussedimmediately above.

Peroxygen compounds that may be used in the disinfection step include,for example, hydrogen peroxide, organic peroxy compounds, and preferablyperacids. Such disinfecting agents are used in a liquid medium,preferably a solution, having a pH of about 1.5 to about 10.0, moredesirably about 2.0 to about 6.0. As to peracid compounds that can beused, these include peracetic acid, perpropioic acid, or perbenzoicacid. Peracetic acid is the most preferred disinfecting agent forpurposes of the present invention.

When used, peracetic acid is desirably diluted into about a 2% to about50% by volume of alcohol solution, preferably ethanol. The concentrationof the peracetic acid may range, for instance, from about 0.05% byvolume to about 1.0% by volume. Most preferably, the concentration ofthe peracetic acid is from about 0.1% to about 0.3% by volume. Whenhydrogen peroxide is used, the concentration can range from about 0.05%to about 30% by volume. More desirably the hydrogen peroxideconcentration is from about 1% to about 10% by volume, and mostpreferably from about 2% to about 5% by volume. The solution may or maynot be buffered to a pH from about 5 to about 9, with more preferredpH's being from about 6 to about 7.5. These concentrations of hydrogenperoxide can be diluted in water or in an aqueous solution of about 2%to about 50% by volume of alcohol, most preferably ethanol. Additionalinformation concerning preferred peroxy disinfecting agents can be foundin discussions in U.S. Pat. No. 6,206,931, which is herein incorporatedby reference.

Flowable ECM materials of the present invention can be prepared to havedesirable properties for handling and use. For example, fluidized ECMhydrolysates can be prepared in an aqueous medium, which can thereafterbe effective to form of a gel. Such prepared aqueous mediums can haveany suitable level of ECM hydrolysate therein. Typically, the ECMhydrolysate will be present in the aqueous medium at a concentration ofabout 2 mg/ml to about 200 mg/ml, more typically about 20 mg/ml to about200 mg/ml, and in some embodiments about 30 mg/ml to about 120 mg/ml. Inpreferred forms, the aqueous ECM hydrolysate composition will have aninjectable character, for example by injection through a needle having asize in the range of 18 to 31 gauge (internal diameters of about 0.047inches to about 0.004 inches).

Furthermore, flowable ECM compositions can be prepared so that inaddition to neutralization, heating to physiologic temperatures (such as37° C.) will substantially reduce the gelling time of the material. Itis contemplated that commercial products or systems of the invention andfor practice of treatments of the invention may include (i) packaged,sterile powders which can be reconstituted in an acidic medium to form aflowable ECM composition, e.g. one which gels when neutralized andpotentially heated; (ii) packaged, sterile aqueous compositionsincluding solubilized ECM hydrolysate components under non-gelling (e.g.acidic) conditions; or (iii) packaged, sterile ECM gel compositions.

In certain embodiments, a medical kit for performing a treatment of avascular vessel in accordance with the invention is provided thatincludes a packaged, sterile aqueous composition including solubilizedECM hydrolysate components under non-gelling (e.g. acidic) conditions,and a separately packaged, sterile aqueous neutralizing composition(e.g. containing a buffer and/or base) that is adapted to neutralize theECM hydrolysate for the formation of a gel. In another embodiment, amedical kit for treatments of the invention includes a packaged,sterile, dried (e.g. lyophilized) ECM hydrolysate powder, a separatelypackaged, sterile aqueous acidic reconstituting medium, and a separatelypackaged sterile, aqueous neutralizing medium. In use, the ECMhydrolysate powder can be reconstituted with the reconstituting mediumto form a non-gelled mixture, which can then be neutralized with theneutralizing medium. The thus neutralized flowable ECM material can thenbe delivered to a treatment site in accordance with the invention beforeor after it gels.

Medical kits as described above may also include a device, such as asyringe, for delivering the neutralized ECM hydrolysate medium to apatient. In this regard, the sterile, aqueous ECM hydrolysate medium orthe sterile ECM hydrolysate powder of such kits can be provided packagedin a syringe or other delivery instrument. In addition, the sterilereconstituting and/or neutralizing medium can be packaged in a syringe,and means provided for delivering the contents of the syringe into amixing container or another syringe containing the aqueous ECMhydrolysate medium or the ECM hydrolysate powder for mixing purposes. Instill other forms of the invention, a self-gelling aqueous ECMhydrolysate composition can be packaged in a container (e.g. a syringe)and stable against gel formation during storage. For example, gelformation of such products can be dependent upon physical conditionssuch as temperature or contact with local milieu present at animplantation site in a patient. Illustratively, an aqueous ECMhydrolysate composition that does not gel or gels only very slowly attemperatures below physiologic temperature (about 37° C.) can bepackaged in a syringe or other container and potentially cooled(including, for example frozen) prior to use for injection or otherimplantation into a patient for a treatment in accordance with theinvention.

In particular applications, ECM hydrolysate compositions that formhydrogels at or near physiologic pH and temperature will be preferredfor in vivo bulking applications, for example in the treatment of stressurinary incontinence, gastroesophageal reflux disease, cosmetic surgery,vesico urethral reflux, anal incontinence and vocal cord repair. Theseforms of the submucosa or other ECM gel have, in addition to collagen,complex extracellular matrix sugars and varying amounts of growthfactors in other bioactive agents that can serve to remodel tissue atthe site of implantation. These ECM hydrolysate compositions can, forexample, be injected into a patient for these applications.

ECM gels and dry sponge form materials of the invention prepared bydrying ECM gels can be used, for example, in wound healing and/or tissuereconstructive applications, or in the culture of cells.

The manipulations used to prepare ECM hydrolysate compositions andgellable or gelled forms thereof can also have a significant impact upongrowth factors or other ECM components that may contribute tobioactivity. Techniques for modulating and sampling for levels of FGF-2,other growth factors, and/or bioactive substances can be used inconjunction with the manufacture of the described ECM hydrolysatecompositions. Illustratively, the dialysis/disinfection processesdescribed above employing peroxy compounds typically cause a reductionin the level of FGF-2 in the ECM hydrolysate material. In work to dateas described in Examples 1-4, such processing using peracetic acid asdisinfectant has caused a reduction in the level of FGF-2 in the rangeof about 30% to about 50%. Accordingly, to retain higher levels ofFGF-2, one can process for a minimal about of time necessary to achievethe desired disinfection of the material; on the other hand, to reducethe FGF-2 to lower levels, the disinfection processing can be continuedfor a longer period of time.

In certain methods of manufacturing a flowable ECM composition, thedisinfection process and subsequent steps will be sufficiently conductedto result in a medically sterile aqueous ECM hydrolysate composition,which can be packaged using sterile filling operations. In othermanufacturing methods, any terminal sterilization applied to the ECMhydrolysate material (e.g. in dried powder, dry sponge form, non-gelledaqueous medium, or gelled form) can also be selected and controlled tooptimize the level of FGF-2 or other bioactive substances in theproduct. Terminal sterilization methods may include, for example, highor low temperature ethylene oxide, radiation such as E-beam, gas plasma(e.g. Sterrad), or hydrogen peroxide vapor processing.

Preferred, packaged, sterilized ECM hydrolysate products for use inaccordance with the invention will have an FGF-2 level (this FGF-2 beingprovided by the ECM hydrolysate) of about 100 ng/g to about 5000 ng/gbased upon the dry weight of the ECM hydrolysate. More preferably, thisvalue will be about 300 ng/g to about 4000 ng/g. As will be understood,such FGF-2 levels can be determined using standard ELISA tests (e.g.using the Quantikine Human Basic Fibroblast Growth Factor ELISA kitcommercially available from R&D Systems).

With reference now to FIGS. 8 and 9, in one illustrative embodiment ofthe invention, all or a portion of a vessel 90 in the region of a defectis surrounded by a flowable composition 94. Flowable composition 94 isdesirably one that promotes the growth of tissue in the patient, and isdesirably a flowable tissue graft material such as a flowable ECMcomposition. In certain embodiments, the flowable composition isinjected about the periphery of the vessel, and allowed or caused to gelor otherwise harden to provide a cuff 96 of material suitable tosupplement or support the vessel wall. Still further, in someembodiments of the invention, the cuff 96 can exhibit retraction orcontraction upon ingrowth of patient tissue, so as to modify the shapeof the vessel in the cuffed region, e.g. to reduce the internal diameterof the vessel. In addition or in the alternative, retraction orcontraction of cuff 96 can increase burst strength and/or elasticity tothe vessel wall. In certain embodiments, the flowable material isintroduced by injection using a cannulated percutaneous device 98 suchas one having needle, which is used to puncture and exit the vessel 90at one or more sites and upon doing so is used to deliver the flowablecomposition through the cannula to surround all or a portion of theexterior of the vessel 90. Alternatively or in addition, an externallydelivered needle 100 can be used to deliver the flowable composition tothe exterior periphery of the vessel 90. The flowable mass, whichoptionally gels or otherwise hardens on its own or can be induced to gelor harden, can serve to reinforce the walls of the vessel 90 and/or toapply pressure to and re-shape the walls of the vessel. In certainembodiments, the flowable mass can be a remodelable ECM biomaterial oranother material that promotes tissue ingrowth. In still furtherembodiments, such tissue ingrowth can lead to retraction or shrinkage ofthe introduced mass as it is replaced by host tissue, thus serving topromote close contact of the ingrown tissue with the vein wall and/or tore-shape the vein wall and potentially modify the function of theinterior valve 92.

FIG. 10 provides an illustration of a vessel 110 and wall portion 114into which a therapeutic composition 116 has been introduced.Composition 116, may, for example, be effective to promote tissuegrowth, and may be a flowable biomaterial as discussed herein.Composition 116 can be delivered through a percutaneous needle device118, which can be manipulated to one or multiple positions (see phantom)to deliver composition 116 into the vessel walls 114. Composition 116and potential tissue development stimulated by composition 116 can serveto strengthen vessel walls 114, for example to protect against initialor continued distension. In addition or alternatively, localizedretraction of vessel walls 114 stimulated by the introduction ofcomposition 116 can serve to re-shape the vessel walls, for example toimpact and improve upon the elasticity and reduce the risks to aneurysmrupture.

The prosthesis and biomaterial either as a construct or as a fluidizedor flowable substance can optionally be rendered radiopaque in whole orin part by a variety of methods as discussed in WO 00/32112, which isincorporated herein. In one embodiment for the invention, when thebiomaterial is formed into sheets whether in lyophilized ornon-lyophilized form a radiopaque substance including but not limitedto, tantalum such as tantalum powder, can be spread along the surface ofthe layers. When the prosthesis or biomaterial is provided as a flowablecomposition, the radiopaque substance can be combined either with thepowdered form or as the hydrated/rehydrated form prior to or duringtreatment. Other radiopaque agents for use in this invention includebismuth, iodine, and barium as well as other conventional markers. Itwill be understood that the radiopaque substance may be incorporatedhomogeneously or inhomogeneously within or on the biomaterial to beimplanted.

Embodiments of the invention have been discussed above generally inconnection with treating vascular vessels at regions associated withdefects. In other embodiments, the inventive methods, constructs andmaterials as described above can be used in the strengthening and/orreinforcement of vascular vessel walls in regions proximate to anintroduced framed or frameless artificial vascular valve. As well, suchstrengthening or reinforcement techniques can be used in conjunctionwith other cut-down surgical or percutaneous treatments to improve anexisting native valve, for example by manipulating or modifying thevalve leaflets themselves. Certain embodiments of the invention,accordingly, involve patient treatment regimens which include treating anative valve or implanting an artificial valve in a vascular vessel incombination with strengthening or reinforcing walls of the vessel inaffected or potentially affected regions associated with the treated orimplanted valve. In more preferred embodiments, the vascular vessel is avein, and the valve is a venous valve. In especially preferred aspects,veins within the legs or feet will be treated in accordance with theinvention.

The invention also encompasses medical products for use in treatments ofthe invention, which products include a prosthesis device or flowablebiocompatible material sealed within sterile medical packaging. Thefinal, packaged product is provided in a sterile condition. This may beachieved, for example, by gamma, e-beam or other irradiation techniques,ethylene oxide gas, or any other suitable sterilization technique, andthe materials and other properties of the medical packaging will beselected accordingly.

In order to promote a further understanding of the present invention andits features and advantages, the following specific examples areprovided. It will be understood that these examples are illustrative andare not limiting of the invention.

EXAMPLE 1

Raw (isolated/washed but non-disinfected) porcine small intestinesubmucosa was frozen, cut into pieces, and cryoground to powder withliquid nitrogen. 50 g of the submucosa powder was mixed with one literof a digestion solution containing 1 g of pepsin and 0.5 M acetic acid.The digestion process was allowed to continue for 48-72 hours underconstant stirring at 4° C. At the end of the process, the digest wascentrifuged to remove undigested material. The acetic acid was thenremoved by dialysis against 0.01 M HCl for approximately 96 hours at 4°C. The resulting digest was transferred (without concentration) into asemipermeable membrane with a molecular weight cut off of 3500, anddialyzed for two hours against a 0.2 percent by volume peracetic acid ina 5 percent by volume aqueous ethanol solution at 4° C. This step servedboth to disinfect the submucosa digest and to fractionate the digest toremove components with molecular weights below 3500. The PAA-treateddigest was then dialysed against 0.01 M HCl for 48 hours at 4° C. toremove the peracetic acid. The sterilized digest was concentrated bylyophilization, forming a material that was reconstituted at about 30mg/ml solids in 0.01 M HCl. This material was neutralized with phosphatebuffered NaOH to a pH of about 7.5-7.6 which provided a flowablematerial which formed a gel when heated to physiologic temperature.

EXAMPLE 2

A second acetic acid processed submucosa gel was made using a processsimilar to that described in Example 1 above, except concentrating thedigest prior to the PAA treatment. Specifically, immediately followingthe removal of acetic acid by dialysis, the digest was lyophilized todryness. A concentrated paste of the digest was made by dissolving apre-weighed amount of the lyophilized product in a known amount of 0.01M HCl to prepare a mixture having an ECM solids concentration of about50 mg/ml. The concentrated paste was then dialysed against the PAAsolution for 2 hours and then against 0.01 M HCl for removal of PAA inthe same manner described in Example 1. The digest was adjusted to about30 mg/ml solids and neutralized with phosphate buffered NaOH to a pH ofabout 7.5-7.6, which upon heating to physiologic temperature formed agel.

EXAMPLE 3

An HCl processed submucosa gel was made using a procedure similar tothat described in Example 1, except using 0.01 M of HCl in thepepsin/digestion solution rather than the 0.5 M of acetic acid, andomitting the step involving removal of acetic acid since none waspresent. The digest was used to form a gel as described in Example 1.

EXAMPLE 4

Another HCl processed submucosa gel was made using a procedure similarto that described in Example 2, except using 0.01 M of HCl in thepepsin/digestion solution rather than the 0.5 M of acetic acid, andomitting the step involving removal of acetic acid since none waspresent. The digest was used to form a gel as described in Example 2.

The present invention contemplates other modifications as would occur tothose skilled in the art. It is also contemplated that methodologiesembodied in the present invention can be altered or added to otherprocesses as would occur to those skilled in the art without departingfrom the spirit of the present invention. All publications, patents, andpatent applications cited in this specification are herein incorporatedby reference as if each individual publication, patent, or patentapplication was specifically and individually indicated to beincorporated by reference and set forth in its entirety herein.

Further, any theory of operation, proof, or finding stated herein ismeant to further enhance understanding of the present invention and isnot intended to make the scope of the present invention dependent uponsuch theory, proof, or finding.

1. A method of treating a patient to modify a vascular vessel having adefect, the method comprising: accessing a treatment site proximate to adefect in a vascular vessel; and introducing a remodelable biomaterialinto a vessel wall or externally of a vessel wall at the treatment site.2. The method of claim 1 wherein the defect is an aneurysm.
 3. Themethod of claim 1 wherein said accessing comprises inserting acannulated delivery device intra-lumenally through the vascular vessel.4. The method of claim 1 wherein said remodelable biomaterial isangiogenic.
 5. The method of claim 3 wherein said introducing comprisesinjecting the remodelable biomaterial through the cannulated deliverydevice into the vessel wall.
 6. The method of claim 3 comprisingextending the cannulated delivery device through the vessel walladjacent the treatment site to deliver the remodelable biomaterial to anexternal surface of the vessel wall.
 7. The method of claim 1 whereinsaid accessing comprises inserting a cannulated delivery deviceexo-lumenally to the treatment site.
 8. The method of claim 7 comprisinginjecting the remodelable biomaterial through the cannulated deliverydevice into the vessel wall.
 9. The method of claim 7 comprisingdelivering the remodelable biomaterial to an external surface of thevessel wall.
 10. The method of claim 1 comprising injecting theremodelable biomaterial into two or more sites spaced circumferentiallyabout the vascular vessel.
 11. The method of claim 1 wherein saidintroducing comprises wrapping a sheet of the remodelable biomaterial incontact with the external connective tissue of the vascular vessel. 12.The method of claim 11 wherein the remodelable biomaterial is a laminatecomprising two or more sheets of a collagenous material.
 13. The methodof claim 11 comprising attaching the sheet of remodelable biomaterial toitself to form a band about the external connective tissue of thevascular vessel.
 14. The method of claim 1 wherein said introducingcomprises delivering an effective amount of the remodelable biomaterialexternally of the vascular vessel to reinforce the vascular vessel wall.15. The method of claim 1 wherein said introducing comprises deliveringthe remodelable biomaterial into the wall or external of the vascularvessel to reshape the vascular vessel.
 16. The method of claim 1 whereinthe remodelable biomaterial is a laminate comprising two or more sheetsof a collagenous material.
 17. The method of claim 1 wherein theremodelable biomaterial comprises an extracellular matrix material. 18.The method of claim 1 wherein the remodelable biomaterial comprisessubmucosa tissue.
 19. The method of claim 1 wherein the remodelablebiomaterial comprises collagen.
 20. The method of claim 18, wherein saidsubmucosa is porcine, bovine, or ovine submucosa.
 21. The method ofclaim 1 wherein the remodelable biomaterial comprises a bioactivesubstance.
 22. The method of claim 1 wherein the remodelable biomaterialcomprises a population of viable cells.
 23. The method of claim 22wherein the cells comprise autologous cells.
 24. A method of treating apatient with a vascular defect, said method comprising: introducing adelivery device through the patient's vasculature to a treatment siteproximate to the vascular defect; and delivering a remodelablebiomaterial into or externally of a wall of the vessel at the treatmentsite.
 25. The method of claim 24 wherein the defect is an aneurysm. 26.The method of claim 24 wherein the defect is a fistula.
 27. The methodof claim 24 wherein said introducing comprises introducing a deliverydevice through a vein.
 28. The method of claim 24 wherein saidintroducing comprises introducing a delivery device through an artery.29. The method of claim 24 wherein said introducing comprises injectingthe remodelable biomaterial through the delivery device into the vesselwall.
 30. The method of claim 24 comprising extending the deliverydevice through the vessel wall to deliver the remodelable biomaterial toan external surface of the vessel wall.
 31. The method of claim 24wherein said delivering comprises delivering an effective amount of theremodelable biomaterial externally of the vascular vessel to reinforcethe vascular vessel wall.
 32. The method of claim 24 wherein saiddelivering comprises delivering the remodelable biomaterial into thewall or externally of the vascular vessel to reshape the vascularvessel.
 33. The method of claim 24 wherein the remodelable biomaterialcomprises an extracellular matrix material.
 34. The method of claim 24wherein the remodelable biomaterial comprises submucosa tissue.
 35. Themethod of claim 24 wherein the remodelable biomaterial comprisescollagen.
 36. The method of claim 35, wherein said submucosa is porcine,bovine, or ovine submucosa.
 37. The method of claim 24 wherein theremodelable biomaterial comprises a bioactive substance.
 38. The methodof claim 24 wherein the remodelable biomaterial comprises a populationof viable cells.
 39. The method of claim 38 wherein the cells compriseautologous cells.
 40. A method for treating a vascular vessel in apatient comprising introducing a remodelable biomaterial into orexternally of a wall of the vessel in a region proximate to an aneurysmor other wall defect.
 41. The method of claim 40 wherein saidremodelable biomaterial has a flowable state, and said introducingcomprises injecting the remodelable biomaterial while in said flowablestate.
 42. The method of claim 41 wherein the remodelable biomaterialalso has a hardened state, and wherein said method also comprisesallowing or causing the remodelable biomaterial to change from saidflowable state to said hardened state after said injecting.
 43. Themethod of claim 42 wherein said remodelable biomaterial is effective tobe thermally induced to change from said flowable state to said hardenedstate.
 44. The method of claim 41 comprising injecting the remodelablebiomaterial into two or more sites spaced circumferentially about thevascular vessel.
 45. The method of claim 41 wherein said injectingcomprises injecting the remodelable biomaterial so as to substantiallysurround the vascular vessel.
 46. The method of claim 40 wherein theremodelable biomaterial comprises a fluidized extracellular matrixmaterial.
 47. The method of claim 40 wherein the remodelable biomaterialcomprises at least one extracellular matrix material selected from thegroup consisting of pericardium, submucosa, liver basement membrane,dura mater, peritoneum, serosa, and renal capsule.
 48. The method ofclaim 40, also comprising introducing a population of viable cells withthe remodelable biomaterial.
 49. The method of claim 48 wherein thecells comprise autologous cells.
 50. The method of claim 40 wherein theremodelable biomaterial comprises a bioactive agent.
 51. The method ofclaim 40 wherein said introducing an amount of the remodelablebiomaterial sufficient to reinforce the vessel wall.
 52. The method ofclaim 40 wherein said introducing an amount of the remodelablebiomaterial sufficient to re-shape the vessel wall.